By way of background, particles can be manipulated by subjecting them to travelling electric fields. Such travelling fields are produced by applying appropriate voltages to microelectrode arrays of suitable design. The microelectrodes have the geometrical form of parallel bars, which may be interrupted by spaces to form channels, as shown in FIG. 1 and may be fabricated using standard metal sputtering and photolithographic techniques as described by Price, Butt and Pethig, Biochemica et Biophysica, Vol.964, pp.221-230. Travelling electric fields are generated by applying voltages of suitable frequency and phases to the electrodes as described in a paper, title "Separation of small particles suspended in liquid by nonuniform travelling field", by Masuda, Washizu and Iwadare, IEEE Transactions on Industry Applications, Vol. IA-23, pp.474-480. Masuda and his coworkers describe how a series of parallel electrodes (with no channels) supporting a travelling electric field can, in principle, be used to separate particles according to their electrical charge and size (weight). Masuda et al have not however described a practical demonstration of such a particle separation method.
In a paper entitled "Travelling-wave dielectrophoresis of microparticles" by Hagedorn, Fuhr, Muller and Gimsa (Electrophoresis, Vol.13, pp.49-54) a method is shown for moving dielectric particles, like living cells and artificial objects of microscopic dimensions, over microelectrode structures and in channels bounded by the electrodes. The travelling field was generated by applying voltages of the same frequency to each electrode, with a 90.degree. phase shift between neighbouring electrodes.
In "Electrokinetic behaviour of colloidal particles in travelling electric fields: Studies using Yeast cells" by Y Huang, X-B Wang and R Pethig J. Phys. D. Appl. Phys. 26 1993 1528-1535, an analysis supported by experiment is made of the "travelling-wave dielectrophoresis" (TWD) effect described by Hagedorn et al (paper cited above). The phenomenological equation ##EQU1## is developed by Huang et al, to show that the TWD velocity is a function of the square of the particle radius (r), the square of the electric field strength (A(0)), the periodic length of the travelling field (.lambda.), medium viscosity (.eta.) and the imaginary part of the Clausius-Mossotti factor f(.epsilon..sub.p *,.epsilon..sub.m *) defining the dielectric properties of the particle and the suspending medium in terms of their respective complex permittivities .epsilon..sub.p * and .epsilon..sub.m *. This equation provides, for the first time, a practical guide for the design of travelling wave electrode systems for the manipulation and separation of particles.
Although the phenomenon in question is usually termed "travelling wave dielectrophoresis", we have now demonstrated that this is something of a misnomer as the force which acts on the particles to produce translational movement is not the dielectrophoresis force but rather that which acts in electrorotation. This force is related to the imaginary component of the polarizability of the particle within its surrounding medium. However, as is discussed in more detail below, particle migration only occurs for travelling wave frequencies which produce negative dielectrophoretic forces on the particle. (Dielectrophoretic forces are related to the real component of the polarizability of the particle within its surrounding medium.) These forces are responsible for lifting the particle away from the electrodes and the channel between the electrodes. We accordingly prefer to refer to the phenomenon called previously "travelling wave dielectrophoresis" by the name "travelling wave field migration" (TWFM). We have established that to obtain TWFM, two separate criteria have to be met. First, a frequency must be selected at which the dielectrophoresis force acting on the particles is negative, i.e. such as to repel the particles from the electrodes. This, we have found requires the real component of the dipole moment induced in the particles to be negative.
Second, the frequency selected has to be such that the imaginary component of the dipole moment induced in the particles is non-zero (whether positive or negative) to produce a force displacing the particles along the array of electrodes.
The present invention is based upon the observation that the TWFM characteristics of a particle (i.e. the direction and speed at which it moves under TWFM and the conditions including electrode layout and spacing, voltage, frequency and suspending medium under which TWFM is possible) can be altered by a selection of methods which affect the dielectric characteristics of the particle concerned.
The present invention provides a method of analysis or separation comprising treating a particle to form an altered particle, which altered particle has TWFM properties distinct from those of the original particle, and producing translational movement of the altered particle by TWFM using conditions under which the movement of the altered particle is different from that which would be obtained using the original particle under identical conditions.
The particle may be of a size to be visible using a light microscope (a microscopic particle) or may be smaller (a sub-microscopic particle) and may be detected using labels such as luminescent, fluorescent and electromagnetic radiation absorbent labels.
Examples of the former type of particles include mammalian cells, plant cells, yeast cells, plastics microbeads, chromosomes undergoing meiosis and mitosis and oocytes, e.g. of Cryptosporidium.
Examples of the second type would include bacterial cells, viruses, DNA or RNA molecules, proteins, other biomolecules, and chromosomes.
The nature of the treatment used to convert the original particle into an altered particle can vary widely according to the nature of the particle. The treatment may involve forming a complex between the particle and a ligand. In some cases, the complex may involve a linking moiety connecting the particle and the ligand. The complex may further include a label connected to the ligand, optionally via a second linking moiety. The complex may involve numerous ligands bound to the particle.
The choice of linking moiety will obviously depend on the nature of the particle and the ligand. For instance if one wishes to capture a nucleic acid species (the ligand) on a plastics micro-sphere (the particle), the linking moiety will normally be chosen to be a nucleic acid or nucleic acid analogue oligomer having a sequence complementary to that of the ligand or a part thereof.
The linking moiety may be bound first to the particle and may then be a species having an affinity for the ligand. Preferably, the affinity for the ligand is a selective affinity such that the formation of the complex between the particle and the ligand is selective and provides at least a degree of identification of the ligand. Preferably, the affinity is highly specific and accordingly the linking moiety bound to the particle which provides the selective affinity for the ligand may be an antibody or an antibody fragment having antibody activity, an antigen, a nucleic acid probe or a nucleic acid analogue probe having selective affinity for complementary nucleic acid sequences, or avidin or an avidin-like molecule such as strept-avidin.
Antibodies and antibody fragments having antibody properties are particularly preferred. There are known techniques suitable for coating antibodies on to the surface of particles such as plastics micro-beads which are well known to those skilled in the art. Antibody coated particles are capable of recognizing and binding corresponding antigens which may be presented on micro-organism cells or some other ligand.
Methods are also known for binding oligo-nucleic acid probes to such micro-beads. Suitable techniques are by way of example described in PCT Application No. GB92/01526, which was published Mar. 4, 1993 under No. WO 93/04199. Where the linking moiety is a nucleic acid probe or a nucleic acid analogue probe, the resulting particle will of course be suitable for recognizing and binding complementary nucleic acid sequences.
The ligand may be chosen to increase the visibility of the particle or otherwise improve its detectability as well as to alter its TWFM characteristics. For instance antibodies bearing fluorophores or chromaphores may be bound to the particle so that the complex so formed can be distinguished from the starting particle by TWFM and detected by fluorescence or colour.
The label may be bound to the ligand either before, simultaneously with, or after the formation of the complex between the ligand and the particle. The label may comprise a second linking moiety carried by the label. Once again, it is preferred that the affinity for the ligand possessed by the second linking moiety is selective, preferably highly specific and the second linking moiety may also be an antibody, an antibody fragment having antibody activity, an antigen, a nucleic acid probe, a nucleic acid analogue probe, avidin or an avidin-like molecule. The use of a label of this nature may be desired to aid ready detection of the complex and/or where a complex between the particle and the ligand does not in itself possess sufficiently distinctive TWFM properties, thus the TWFM may be further altered by the inclusion in the complex of the label. To this end, the label may be a fluorophore or chromaphore, or a micro-organism, a metal particle, a polymer bead or a magnetic particle. For use in connection with TWFM measurements, the label preferably has dielectric properties and is capable of acquiring a significant surface charge. A particularly preferred material is colloidal gold which is easily bound to antibodies (as the second species) to form a label. Antibodies bound to colloidal gold are commercially available and methods for binding antibodies to colloidal gold are for instance described in Geohegan W. D. et al (1978) Immunol. Comm 7 pl. Other metal particles however may be employed, e.g. silver particles and iron particles.
The use of a label of the kind described above may be preferred even where a complex between the ligand and a particle possesses sufficiently distinctive TWFM properties to enable the formation of such a complex to be observed. A higher level of specificity may in certain cases be obtained by the use of a label in such a complex. Thus for instance, one may wish to distinguish a micro-organism expressing an antigen A from a micro-organism expressing antigens A and B. This may be accomplished by the use of micro-particles having as a linking moiety an antibody to A and a label having as it's moiety an antibody to B. The difference in the velocities of the labelled complex (between the micro-particle, the micro-organism and the label) and the unlabelled complex (between the micro-particle and the micro-organism) can be observed, and used to distinguish micro-organisms expressing antigen A only, from those expressing A and B.
The label may include a magnetic particle so that the label can be attracted to a magnet so as to concentrate complexes containing the label for easier observation. In some cases it may be possible to attract labelled complexes to a magnet and to wash away unlabelled particles so as to eliminate the background of particles bearing linking moieties but no ligand/label, which would normally be present. Suitable magnetic labels for this purpose will include iron micro-particles bearing linking moieties such as antibodies. Such antibody coated iron particles are commercially available.
Labels for both cells and smaller particles can include fluorescent markers, e.g. FITC or rhodamine, chromophores, luminescent markers or enzyme molecules which can generate a detectable signal. Examples of the latter include luciferases and alkaline phosphatase. These markers may be detected using spectroscopic techniques well known to those skilled in the art. The label could be bound to the ligand either before, simultaneously with, or after the formation of the complex between the ligand and the particle. In the case of cell transfection, the cells may co-express a marker with the gene product. For example, the gene for firefly or bacterial luciferase may be co-transfected into the cells enabling the transfected cells to be visualized by a luminescent signal.
The alteration of the original particle need not involve the formation of a complex. For instance, the TWFM characteristics of a cell may be altered by heating or by treatment with a reagent which alters the porosity of the cell membrane. Accordingly, the alteration may be in the particle itself rather than or in addition to a change due to the physical presence of a ligand in a complex. Both effects may be present in combination. A ligand forming a complex with a particle may exert a physical effect on the TWFM properties and also by interaction with the particle bring about a change in the intrinsic TWFM properties of the particle.
The invention includes methods as described above carried out for analytical purposes and also such methods carried out for preparative or other purposes.
The methods according to the invention may be employed in a wide variety of analytical applications including separation and analysis of samples containing cells for example, bacterial, mammalian, yeast, and insect cells or virus particles, and, biological macromolecules. Current methods of separating cells, for example flow cell cytometry, require expensive instrumentation, skilled operators and significant laboratory resources. The techniques also have limitations when there are many different cell populations to be separated and when the cells of interest represent less than a few percent of the total. For separation and analysis of modified biological molecules, or complexes between biological macromolecules, employed techniques include electrophoresis and chromatographic separation using gel-filtration or affinity chromatography. Although these, in some cases, provide adequate separation, for many applications they can be time consuming and have limited resolution. In addition, use of these methods can affect the equilibrium between biological complexes. For example, gel-filtration results in a significant dilution of the sample.
Methods described hereafter according to the present invention allow some or all of these drawbacks to be addressed.
Where in an analytical method according to the invention a complex between the particle and a ligand is produced, the ligand need not itself be the species to establish the presence, nature or quantity of which is the ultimate purpose of the analysis. Thus, the ligand may be a reagent in the analysis and the species of interest in the analysis may be another component of the complex, e.g. the linking moiety or the particle itself. Where a particle is altered by treatment with a reagent, it may be the particle or the reagent which is essentially to be studied.
The process of TWFM described previously has been carried out using an array of linear, parallel electrodes subjected to phased electric fields normally such that every fourth electrode along the TWFM path is in phase. This periodicity defines the effective wave length of the travelling wave field produced. We have established that this wave length is optimally about ten times the average diameter of the particle to be moved under TWFM, e.g. from 5 to 20 times or more preferably 8 to 12 times said average diameter. For particles which are not roughly circular, it is the length in the direction transverse to TWFM movement which is of significance.
The electrodes may be formed, depending on the dimensions required, using any of the standard techniques for patterning and manufacturing microscopic structures. For example the electrodes can be produced by:
screen printing; PA1 deposition of electrode material (e.g. by electroplating or sputter deposition) followed by one of the following patterning techniques: PA1 patterning followed by deposition of the electrode material (as in the X-ray LIGA process).
direct writing using an electron beam followed by etching (e.g. wet chemical etching, dry plasma etching or focused ion beam etching); PA2 writing by exposure through a photolithographically generated mask followed by etching--the mask may be generated for example by visible, ultra violet, X-ray or electron beam lithography; PA2 excimer laser ablation;